In this study, we investigated the mechanism of deregulation of TGFβ signaling in chronic VUs. The TGFβ signaling cascade is attenuated by downregulated expression of all three major receptors, loss of Smad2 activation, and deregulation of TGFβ target genes ().
Figure 5 Attenuation of TGFβ signaling in VU. Top, histology of healthy skin. Cartoon summarizes a simplified signaling cascade in healthy epidermis. Bottom, histology of VU. In addition to decreased levels of TGFβ , downregulation of receptors (more ...)
Based on the therapeutic effect of topical application in animal models (50
), TGFβ has been a promising therapeutic option for impaired wound healing. In rats and humans with impaired wound healing, overall TGFβ levels in wound fluid were shown to be diminished, and the normal elevation of TGFβ 1 found during acute wound healing was absent (21
). Furthermore, differential expression of the TGFβ receptors in acute and chronic wounds has been described ((3
)). However, TGFβ 1 failed to get into clinical trials (29
). Our data demonstrate that TGFβ signaling is further impaired by reduced expression of the TGFβ receptors in the wound edge of VU in addition to decreased TGFβ levels. This impaired signaling leads to an abrogation of Smad2 activation and deregulation of TGFβ target genes. Functional loss of the TGFβ /Smad signaling cascade in VU offers an explanation for the limited ability of exogenous applications of TGFβ to accelerate wound healing in chronic wounds.
An important study analyzing TGFβ receptors using qPCR on biopsies of human nonhealing VUs has suggested that the absence of TGFβ RII contributes to the chronicity of these ulcers (20
). The study also looked at TGFβ RI and found high expression throughout the thickened epidermal margin of the ulcers, including the suprabasal layers, and within fibroblasts in the dermal ulcer margin (20
). In contrast, we found a complete absence of TGFβ RI and TGFβ RII and downregulation of TGFβ RIII. These differences in findings might be attributed to the wound location from which biopsies were taken. To assure that biopsies obtained indeed originated from non-healing tissue, we used a previously established marker (nuclearization of β-catenin) that demarcates nonhealing tissue within chronic wounds (30
Inhibition of keratinocyte mitosis is an important step in the early stages of acute wound healing, when keratinocytes are being recruited to the wound edge to migrate and properly epithelialize the wound site (52
). In addition to its role during cutaneous wound healing, TGFβ signaling plays a profound role in maintaining epithelial tissue homeostasis by suppressing proliferation (7
). Because of this important role we focused on the TGFβ pathway in VUs compared with healthy skin and found suppression of signaling components. Our data suggest that attenuation of TGFβ signaling in VUs could contribute to the hyperproliferative phenotype of nonhealing epidermis (2
). TGFβ responses include repression of growth-promoting transcription factors, most notably c-myc (53
). This finding is in agreement with our previous data that showed elevated c-myc expression and the presence of β-catenin in the nucleus at the non-healing wound edge of VUs (30
). Thus, we conclude that the lack of TGFβ signaling might contribute to c-myc over -expression and loss of cytostatic control in nonhealing VU epidermis (). The transcription factors we found suppressed in VUs, GADD45β and ATF3 are also known to participate in TGFβ-mediated growth control in epithelial cells (44
). ATF3 is also a stress response gene that can be activated by cellular stresses and mechanical injury (56
). Its suppression in VUs suggests that epidermis in chronic wounds may be unresponsive to both stress and growth factor therapy.
Loss of responsiveness to TGFβ is a hallmark of many types of cancers (54
). Although carcinomas are documented to arise at the site of chronic wounds, they are a relatively infrequent complication (59
). Similar to chronic wounds, conditional knockout of TGFβ RII in stratified epithelia led to infrequent and localized tumor development only in older animals (60
). Strong induction of CSTA in VUs may be an additional factor in providing protection against malignant transformation in VUs, because CSTA is reduced in skin cancers (61
The role of Smad7, an inhibitor of the Smad signaling cascade, has not yet been studied in chronic wounds. Our study demonstrated profound downregulation of Smad7 in the basal layer of the epidermis of VUs, whereas Smad7 was upregulated in keratinocytes of acute wounds ex vivo
. The human skin organ culture model has been extensively and reliably used to study epidermal wound healing ((33
)); however, it was never used to analyze TGFβ signaling. Showing induction of Smad2 phosphorylation and up-regulation Smad7, we confirmed activation of TGFβ signaling in an acute ex vivo
wound model. In a mouse model of acute wound healing, increased levels of Smad7 coincided with delayed reepithelialization of wounds, whereas downregulation of Smad7 accelerated reepithelialization (63
). In contrast, we observed reduced expression of Smad7 in the basal epidermis of nonhealing wounds in parallel to reduced levels of pSmad2 and deregulated target gene expression. Thus, downregulation of Smad7 did not rescue TGFβ-signaling activity via
the Smad cascade. This finding indicates that Smad7 is not responsible for the inhibition of TGFβ signaling in VUs, but instead the strong decrease in TGFβ receptor levels causes a functional knockdown of TGFβ signaling.
In summary, this study demonstrates that in the nonhealing wound edge of VUs, TGFβ signaling is attenuated by decreased expression of receptors, resulting in diminished activation of the Smad signaling cascade and subsequent deregulation of TGFβ target genes leading to loss of tissue homeostasis and hyperproliferation. Our results provide strong evidence that disruption of the TGFβ signaling cascade may be the underlying factor for failure of exogenous TGFβ to accelerate healing in chronic wounds.